The long-term goal of this research is to study the physical determinants of outer hair cell electromotility. The outer hair cell enhances the sensitivity and frequency selectivity of mammalian hearing by converting the energy of intracochlear electrochemical gradients into mechanical energy. The motor mechanism responsible resides in the outer hair cell's lateral wall, a 100 nanometer thick, three-layer structure composed of two membranes with a cytoskeletal network sandwiched between them. Energy conversion by the lateral wall is bidirectional (electrical to mechanical and mechanical to electrical). The tight coupling between electrical polarization and mechanical displacement is characteristic of piezoelectricity. The specific objectives of this project period are to 1) identify the mechanisms that contribute to the high frequency membrane potentials that drive electromotility and 2) to identify the contributions of the lateral wall to the modulation and maintenance of the electrochemical gradients necessary for cell function. Coordinated theoretical and experimental approaches identify the piezoelectric and ionic contributions to the cell's high frequency response. They also address how the unique molecular organization of the lateral wall influence the diffusion of membrane constituents and contribute to the transport of ions, water and other molecules through the narrow space between its membranes. Experiments will assess the role of the membrane protein prestin by recording from outer hair cells isolated from normal and prestin null mutant mice. The lateral mobility of fluorescent lipid analogues and membrane proteins will be determined. Methods include the development of new transgenic mice, micro electro impedance spectroscopy, voltage and current-clamp, including two-pipette recording; fluorescence recovery after photobleaching, confocal microscopy, total internal reflection photolysis, video microscopy and computational modeling. The studies will further clarify the role of the outer hair cell as the cochlear amplifier. Clarification of the physical principles underlying electromotility will also contribute to the emerging field of biological nanotechnology.